The long term goal of this project is to understand the role of tumor suppressor genes in signal transduction pathways in nuclei. Interruption of the signal cascade frequently leads to malignant growth of cells. Mutation of two tumor suppressor genes, RB and p53, results in deregulated cell growth and provides an example of interruption of the pathway. These two genes are frequently found mutated in many human tumor types including breast carcinoma. Recent studies have demonstrated that RB interacts with several cellular proteins. One of the best characterized RB-associated proteins, E2F- 1, is a transcriptional factor that is involved in the regulation of a set of cellular genes involved in G1/S transition. We have isolated genes encoding two other nuclear RB-associated proteins, p48 and p46. These two genes share high sequence homology with a yeast gene, MSI1, that is presumed to be a negative regulator of the Ras signal pathway. We showed that human p48 can substitute for yeast MSI1 in suppressing mutant Ras activities. To address whether p48 and p46 provide a connection between outside signals and nuclear suppressor genes, we propose continuing studies of p48 and p46 in mammalian cells. To establish a mouse mammary gland tumor model reflecting the processes following mutational inactivation of tumor suppressor genes, and address how these mutations affect the response of mammary epithelial cells to physiological signals, we propose to use phage site-specific recombinase Cre for generation of mammary gland-specific mutations of RB and p53. These model systems will allow detailed dissections of changes in the mammary epithelial cells that lead to tumor formation. The following aims are proposed: (A) To study the structural requirements of p48 and p46 for their activities in yeast and their interactions with RB. The expression pattern of p48 and p46 in different tissues will be compared. (B) To assess the function of p48 and p46 in mammalian cells using eukaryotic expression systems. Three cell types, PCc12, C-Ha-ras(Val-12) transformed 3T3 cells, and RB-positive clones derived from breast tumor cell lines harboring RB mutations will be used: (C) To develop silent p53 target vectors for replacing the endogenous gene in mouse embryonic stem cells. This step is essential in order to generate tissue-specific inactivation of the targeted genes. (D) Generation of mice carrying mammary gland-specific mutations of p53 and RB. A series of crosses between Cre-expressing mice and mice carrying silent mutations will be made. The development of mammary tumors in these mice will be followed. Growth and differentiation of the mammary epithelial cells in response to lactogenic signals will be studied in the transgenic animals. How these mammary epithelial cells respond to physiological hormonal signals will be studied in primary cultures.